A major impediment to successful treatment of Staphylococcus aureus is its tendency to develop antibiotic resistance. This organism presents additional complications by establishing biofilm communities, typically at sites of surgical implants and avascular tissue such as heart valves. Biofilms maintain a subpopulation of dormant cells not affected by most antibiotics because they target active metabolic processes. Surgical intervention can remove specific foci of infection but cannot address bacteremia and sepsis that frequently make these infections fatal. Development of systemic, non-antibiotic strategies against S. aureus has become a public health imperative, highlighted recently by the emergence of extraordinarily virulent strains affecting otherwise healthy individuals in schools and other non-hospital settings. The enzyme lysostaphin kills virtually all strains of S. aureus, including methicillin-resistant S. aureus (MRSA). Lysostaphin acts by binding to and cleaving interpeptide bridges in cell wall peptidoglycan, leaving bacteria vulnerable to osmotic lysis. This enzyme is effective against dormant cells, and although lysostaphin resistance occurs, it generally coincides with increased susceptibility to antibiotics. This leads to the belief, demonstrated in mammalian models of Staphylococcal infection, that coadministration of lysostaphin with traditional antibiotics will improve clinical outcomes by clearing S. aureus to below detectable levels faster and more consistently, and dramatically improving survival. Clinical development will require a thorough understanding of lysostaphin's mode of action and immunogenic properties, which is lacking at present. The catalytic and cell-wall targeting mechanisms of lysostaphin are poorly characterized. A C-terminal cell-wall targeting domain binds the catalytic substrate and is required for full activity of the catalytic domain, but the precise requirements for targeting are not known. It is not clear whether the two domains bind simultaneously or sequentially, or whether they bind overlapping or adjacent targets in the cell wall. To resolve these issues, we will solve the three-dimensional structure of lysostaphin by solution-state nuclear magnetic resonance (Aim 1), and characterize binding of peptidoglycan to the intact, active enzyme by chemical shift perturbation and NMR relaxation studies (Aim 2). The results of this work will be an important advance in our knowledge of lysostaphin and similar enzymes, and represent a major step toward developing the clinical utility of this potent tool against a pathogen of enormous consequence to public health. PUBLIC HEALTH RELEVANCE A major impediment to successful treatment of Staphylococcus aureus is its tendency to develop antibiotic resistance. Methicillin-resistant Staphylococcus aureus (MRSA) has become a household word in the U.S., with recent outbreaks occurring among otherwise healthy school students with sometimes fatal consequences. Even without antibiotic resistance, S. aureus can form biofilms that protect dormant cells from antibiotics, most of which work only against active cells. New compounds that work differently than traditional antibiotics are desperately needed to keep ahead of the MRSA epidemic. The enzyme lysostaphin has great potential because it targets both active and dormant cells, and also because when the bacteria become resistant to lysostaphin, they usually also become less resistant to antibiotics. Our work seeks to understand the way lysostaphin targets S. aureus by determining its atomic-resolution structure by nuclear magnetic resonance spectroscopy (NMR) and using that structure to explore regions of the enzyme that make direct contact with the cell-wall of MRSA cells. The results of this work will be an important advance in our knowledge of lysostaphin and similar enzymes, and represent a major step toward developing clinical use of this potent tool against a pathogen of enormous consequence to public health.